The cell autonomous molecular events that drive the initiation and progression of pancreatic cancers are multifarious and complex. This context makes identification of effective intervention and prevention strategies a seemingly daunting task. However, widespread evidence suggests that a unifying principle governing formation of a "minimal oncogenic platform" is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Linchpin proteins recruited to drive these pathways during tumorigenesis likely represent optimal intervention targets in a heterogeneous population of cancer cells. We have established that high-throughput siRNA-mediated loss-of-function analysis can reveal novel linchpin proteins selectively supporting the survival of cancer cells versus normal epithelia. This approach can now be pursued on a genome-wide scale for an unbiased comprehensive analysis of the molecular framework supporting pancreatic cancer cell survival. Here we will combine an in-hand arrayed genome-wide human siRNA library with high-throughput cell-based screening platforms for broad-spectrum identification of optimal genetic targets for focused development of effective chemotherapeutic agents. Our goal is the identification of optimal molecular targets to help guide rational design of novel combinatorial drug-based therapies for human pancreatic cancer. Our immediate objective is to advance this goal by the comprehensive identification of genes that support drug resistance in treatment-refractory human pancreatic cancer cells. Agents that target these gene products or the pathways they regulate will be prime candidates for effective multi-drug therapies. Our approach is to combine a genome-wide siRNA array with a validated high-throughput cell-based screening platform to identify gene targets that expose enhanced-sensitivity to known chemotherapeutic compounds. Our Specific Aims are: 1) To identify molecular targets selectively supporting drug resistance in human pancreatic cell lines, and 2) To validate the effectiveness of candidate targets within an orthotopic xenograft model of pancreatic cancer. The approach we describe here will facilitate the unambiguous identification of linchpin proteins selectively required for survival of pancreatic cancer cells upon exposure to gemcitibine orcyclopamine, two drugs respectively representing the commonly used but less-than-effective current therapy versus an effective agent in preclinical models yet to be tested in humans. This information will 1) guide rational selection of available drugs and drug combinations for development of new intervention and/or prevention strategies, and 2) help identify novel pharmaceutically tractable therapeutic targets whose inhibition will have minimal impact on normal cells. [unreadable] [unreadable] [unreadable]